Insulin-treated Rats Research Articles

Jamun (Syzygium cumini Linn.) Seed Extract as a Potential Antidiabetic Agent: Effects on Blood Glucose Levels, Haematological Parameters and Biochemical Profiles in Alloxan-Induced Diabetic Albino Rats

Diabetes mellitus is rapidly increasing in India, making it essential to find alternative therapies that minimize side effects and improve patient outcomes. Safer treatment options are needed to manage the growing burden of this chronic condition effectively. This study evaluates the antidiabetic potential of Syzygium cumini seed extract (SCSEt) in alloxan-induced diabetic rats. Diabetes was induced by administering alloxan (120 mg/kg) in albino rats, and diabetes was confirmed with blood glucose levels ≥290 mg/dL. Rats were treated with SCSEt at doses of 2, 4, and 6 g/kg orally, with insulin (1.0 IU/kg/day) serving as a reference treatment. Over four weeks, SCSEt demonstrated a significant reduction in blood glucose levels compared to untreated diabetic controls, though less effective than insulin. Blood glucose levels in SCSEt-treated groups decreased from 286 ± 1.10 mg/dL (low-dose) to 110 ± 3.21 mg/dL (high-dose), compared to insulin-treated rats with levels of 112 ± 1.96 mg/dL. Haematological parameters improved with SCSEt, particularly at higher doses, where packed cell volume (PCV) reached 42.91 ± 0.98% and haemoglobin levels were 17.08 ± 2.06 g/dL. Biochemical analyses revealed reduced serum cholesterol and improved protein levels in high-dose SCSEt rats, with cholesterol dropping to 112 ± 1.92 mg/dL and protein rising to 6.39 ± 0.09 mg/dL. SCSEt also enhanced glucose tolerance, with high-dose SCSEt rats achieving the lowest glucose level of 104 ± 1.62 mg/dL at 100 minutes during the oral glucose tolerance test (OGTT). Although SCSEt was less effective than insulin in glucose management, its potential as a complementary therapy is supported by its significant improvements in glycemic control and lipid profile.

Monitoring glucose levels in urine using FTIR spectroscopy combined with univariate and multivariate statistical methods

The development of novel platforms for non-invasive continuous glucose monitoring applied in the screening and monitoring of diabetes is crucial to improve diabetes surveillance systems. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy of urine can be an alternative as a sustainable, label-free, fast, non-invasive, and highly sensitive analysis to detect changes in urine promoted by diabetes and insulin treatment. In this study, we used ATR-FTIR to evaluate the urinary components of non-diabetic (ND), diabetic (D), and diabetic insulin-treated (D + I) rats. As expected, insulin treatment was capable to revert changes in glycemia, 24-h urine collection volume, urine creatinine, urea, and glucose excretion promoted by diabetes. Several differences in the urine spectra of ND, D, and D + I were observed, with urea, creatinine, and glucose analytes being related to these changes. Principal components analysis (PCA) scores plots allowed for the discrimination of ND and D + I from D with an accuracy of ∼ 99 %. The PCA loadings associated with PC1 confirmed the importance of urea and glucose vibrational modes for this discrimination. Univariate analysis of second derivative spectra showed a high correlation (r: 0.865, p < 0.0001) between the height of 1074 cm-1 vibrational mode with urinary glucose concentration. In order to estimate the amount of glucose present in the infrared spectra from urine, multivariate curve resolution-alternating least square (MCR-ALS) was applied and a higher predicted concentration of glucose in the urine was observed with a correlation of 78.9 % compared to urinary glucose concentration assessed using enzyme assays. In summary, ATR-FTIR combined with univariate and multivariate chemometric analyses provides an innovative, non-invasive, and sustainable approach to diabetes surveillance.

A Potential Treatment Strategy for the Treatment of Diabetic Kidney Disease in Streptozotocin-induced Diabetic Rats: Leptadenia Hastata Extract

Background: Kidney disorders are serious outcomes of diabetes, resulting in renal ailments. This study seeks to provide an alternative treatment for the disorder through the use of medicinal plants that offer renal protection and ameliorate the deleterious effect of diabetes. Methods: Thirty rats were divided into six groups of five and hyperglycemia was induced by intraperitoneal injection of 50 mg/kg streptozotocin. The rats with a fasting blood glucose level exceeding 250 mg/dl were selected. The L. hastata leaf extract was administered orally and a daily insulin injection was given intramuscularly into the left thigh for 28 days. Rats were then sacrificed and the urine was collected for urinalysis. The kidneys were harvested and examined histologically. The micrographs obtained were subjected to morphometric analyses to evaluate several parameters. Results: The extract-treated groups showed preservation of the cytoarchitecture of the renal tubules and glomeruli as compared to the diabetic control group, which showed distortion of the glomeruli and atrophied renal tubules. The rats that received the extract showed a significantly increased glomerular perimeters and areas (P&lt;0.01). The cellularity was significantly increased (P&lt;0.001); however, the glomerular basement membranes were significantly thicker in these groups compared to the controls (P&lt;0.001). Both the extract-treated and insulin-treated rats showed reduced urine glucose concentrations. Urobilirubinogen, protein, and ketone levels were elevated in the diabetic controls compared to the extract-treated rats. Conclusion: Administration of the L. hastata extract led to renal protection by preserving the cytoarchitecture of the glomeruli and renal tubules, restoring the kidneys’ function.

9-LB: Increased Islet Expression of Somatostatin and Glucagon in Male Rats with Insulin-Dependent Type 2 Diabetes

Background: Pancreatic islet remodelling in type 1 diabetes increases somatostatin (SST) expression, which inhibits glucagon counterregulation to hypoglycemia. Here, we characterized islet morphology and hormone composition in a rodent model of insulin-deficient type 2 diabetes (T2D) . Methods: Hypoinsulinemic T2D was induced in 8-9-week-old, male, Sprague-Dawley rats (n=4) via 3 weeks of high-fat feeding and a low-dose injection of streptozotocin (STZ; 35 mg/kg) . After 2 weeks of basal insulin treatment, pancreas tissue was extracted from T2D and healthy control (n=5) rats for immunohistochemical staining of representative islets. Results: Fasted plasma C-peptide levels of hyperglycemic (24.8±3.3 mmol/l) T2D rats were reduced by ∼44% (624±64 vs. 1107±121 pmol/l; p&amp;lt;0.001) , while glucagon was unchanged (75±31 vs. 98±39 pg/ml) . Fractional islet area of glucagon and SST were elevated by 40% (21±15 vs. 15±10%; p&amp;lt;0.001) and 83% (11±8 vs. 6±5%; p&amp;lt;0.001) , respectively, as compared to control rats (Figure) , which was most pronounced in small and medium-sized islets. Conclusions: Fractional area expression of glucagon and SST increased per islet in insulin-treated rats with advanced T2D. These findings may help account for the increase in SST signaling that underscores defective glucagon counterregulation to hypoglycemia in insulin-deficient diabetic states. Disclosure E. G. Hoffman: None. S. C. Atherley: None. N. Akbarian: None. N. Dsouza: None. R. Liggins: Employee; Zucara Therapeutics, Stock/Shareholder; Zucara Therapeutics. M. Riddell: Advisory Panel; Zealand Pharma A/S, Zucara Therapeutics, Consultant; Eli Lilly and Company, Jaeb Center for Health Research, Speaker’s Bureau; Dexcom, Inc., Eli Lilly and Company, Novo Nordisk. Funding GlycoNet

Citric Acid Administration Protects Against Insulin-Induced Hypoglycemic Brain and Liver Injury

We aimed to investigate the effect of treatment with citric acid on oxidative stress and tissue injury in brain and liver of rats subjected to hypoglycaemia. Rats received intraperitoneal injections of insulin (5 IU/kg) followed 30 min later by oral administration of saline or citric acid at doses of 0.2, 0.4 or 1 g/kg. Rats were euthanized 1 h after insulin injection and their brains and livers were dissected out for biochemical studies including determination of malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide (NO), paraoxonase-1 (PON-1), 5-lipoxygenase, and cholinesterase. Serum alanine aminotransferase and aspartate aminotransferase were determined and histological investigations for the liver were carried out. Results indicated that in insulin only treated rats, MDA and NO levels were significantly increased whereas GSH content and PON-1 activity significantly decreased in brain and liver tissue. In addition, cholinesterase activity significantly decreased while 5-lipoxygenase increased in brain with respect to the saline control group after insulin injection. Serum aminotransferases showed significant elevation in insulin-treated rats together with distorted hepatic architecture, hydropic degeneration, and extensive cytoplasmic vacuolation of hepatocytes. The biochemical alterations in the brain and liver of hypoglycaemic rats were markedly alleviated in a dose-dependant manner by the administration of citric acid which also markedly reduced the histological liver injury with the group treated with insulin and citric acid at 1 g/kg exhibiting more or less preserved hepatic architecture. These results indicate that citric acid was effective in alleviating the biochemical alterations in brain and liver and protecting against liver injury during hypoglycaemia. It could thus prove of benefit in patients on insulin therapy with accidental hypoglycaemia from over dosing.

Comparative Evaluation of the Effect of Metformin and Insulin on Gut Microbiota and Metabolome Profiles of Type 2 Diabetic Rats Induced by the Combination of Streptozotocin and High-Fat Diet.

Lately, an increasing number of studies have investigated the relationship between metformin and gut microbiota, suggesting that metformin exerts part of its hypoglycemic effect through the microbes. However, its underlying mechanism remains largely undetermined. In the present study, we investigated the effects of metformin on gut microbiota and metabolome profiles in serum and compared it with insulin treatment in rats with type 2 diabetes mellitus (T2DM). Diabetic rats (DM group) were induced by a combination of streptozotocin and high-fat diet (HFD). After 7 days, DM rats were treated with metformin (MET group) or insulin (INS group) for 3 weeks. The 16S rRNA sequencing of the gut microbiota and non-targeted metabolomics analysis of serum were conducted. A total of 13 bile acids (BAs) in serum were further determined and compared among different groups. The rat model of T2DM was well established with the typical diabetic symptoms, showing significantly increased blood glucose, AUC of OGTT, HOMA-IR, TC, TG, LDL-C and TBA. Metformin or insulin treatment could ameliorate symptoms of diabetes and partly recover the abnormal biochemical indicators. Compared with DM rats, the relative abundances of 13 genera were significantly changed after metformin treatment, while only three genera were changed after insulin treatment. The metformin and insulin treatments also exhibited different serum metabolome profiles in T2DM rats. Moreover, 64 differential metabolites were identified between MET and DM groups, whereas 206 were identified between INS and DM groups. Insulin treatment showed greater influence on amino acids, glycerophospholipids/glycerolipids, and acylcarnitine compared with the metformin treatment, while metformin had an important impact on BAs. Furthermore, metformin could significantly decrease the serum levels of CA, GCA, UDCA, and GUDCA, but increase the level of TLCA in DM rats. Insulin treatment significantly decreased the levels of CA, UDCA, and CDCA. Besides, several metabolites in serum or microbiota were positively or negatively correlated with some bacteria. Collectively, our findings indicated that metformin had a stronger effect on gut microbiota than insulin, while insulin treatment showed greater influence on serum metabolites, which provided novel insights into the therapeutic effects of metformin on diabetes.

133-OR: Somatostatin Receptor Type 2 Antagonism (SSTR2a) Augments Glucagon Counterregulation and Delays Hypoglycemia Onset in Type 2 Diabetic (T2D), Sprague-Dawley Rats

Background: SSTR2a rescues glucagon counterregulation in type 1 diabetic rats. Here, we investigated its potential for resisting hypoglycemic onset in insulin-treated rats with T2D.Methods: T2D was induced in 8-9-week-old, male, Sprague-Dawley rats (n=20) via 3-weeks of high-fat feeding and low-dose streptozotocin (35 mg/kg). After 1 week of basal insulin maintenance, N=10 healthy and N=20 T2D rats were treated with SSTR2a (3 mg/kg ZT-01, SC) or vehicle in a crossover design (1 wk apart), 60 min prior to hypoglycemic induction via insulin aspart bolus (6 U/kg).Results: SSTR2a increased plasma glucagon levels for up to 120 min after insulin challenge, with a 2.2-fold increase in glucagon AUC (32167 ± 6658 vs. 14428 ± 3673 pg*min/mL; p<0.01). Blood glucose was significantly elevated in treated, T2D rats for ∼30 min, and hypoglycemia onset was delayed by 17 ± 4 min (p<0.001). Neither effect was observed in SSTR2a-treated, healthy rats.Conclusions: SSTR2a increased glucagon secretion following insulin overdose and delayed the onset of hypoglycemia in T2D rats. SSTR2a may be a promising therapeutic tool for alleviating hypoglycemic risk in intensively-treated T2D.View largeDownload slideView largeDownload slide DisclosureE. G. Hoffman: None. N. Dsouza: None. J. Aiken: None. R. Liggins: Employee; Self; Zucara Therapeutics Inc. M. Riddell: Advisory Panel; Self; Zealand Pharma A/S, Consultant; Self; Lilly Diabetes, Research Support; Self; Dexcom, Inc., Insulet Corporation, Speaker’s Bureau; Self; Novo Nordisk Inc., Sanofi, Stock/Shareholder; Self; Zucara Therapeutics Inc. FundingGlycoNet

Anti-hyperglycaemic effects of dioxidovanadium complex cis-[VO2(obz)py] avert kidney dysfunction in streptozotocin-induced diabetic male Sprague-Dawley rats.

Despite the success of antidiabetic drugs in alleviation of hyperglycaemia, diabetic complications, including renal dysfunction, continue to be a burden. This raises the need to seek alternative therapies that will alleviate these complications. Accordingly, the aim of this study was to investigate the effects of dioxidovanadium(V) complex cis-[VO2(obz)py] on renal function in diabetic rats. Streptozotocin-induced diabetic rats were treated with cis-[VO2(obz)py] (40 mg·kg-1) twice every third day for five weeks. Diabetic untreated and insulin-treated rats served as the diabetic control and positive control, respectively. Blood glucose concentrations, water intake, urinary output, and mean arterial pressure (MAP) were monitored weekly for five weeks. Rats were then euthanized, and blood and kidney tissues were collected for biochemical analysis. Significant decreases in blood glucose concentrations, MAP, glomerular filtration rate (GFR), and SGLT2 expression, as well as plasma angiotensin and aldosterone concentrations, were observed in the treated groups compared with the diabetic control. The complex also increased urinary glucose concentrations, antioxidant enzymes GPx and SOD concentrations, and decreased MDA concentrations and kidney injury molecule (KIM-1) concentrations. These findings suggest that the anti-hyperglycaemic effects of this vanadium complex may ameliorate kidney dysfunction in diabetes.

Cerebral Insulin Bolus Revokes the Changes in Hepatic Lipid Metabolism Induced by Chronic Central Leptin Infusion.

Central actions of leptin and insulin on hepatic lipid metabolism can be opposing and the mechanism underlying this phenomenon remains unclear. Both hormones can modulate the central somatostatinergic system that has an inhibitory effect on growth hormone (GH) expression, which plays an important role in hepatic metabolism. Using a model of chronic central leptin infusion, we evaluated whether an increase in central leptin bioavailability modifies the serum lipid pattern through changes in hepatic lipid metabolism in male rats in response to an increase in central insulin and the possible involvement of the GH axis in these effects. We found a rise in serum GH in leptin plus insulin-treated rats, due to an increase in pituitary GH mRNA levels associated with lower hypothalamic somatostatin and pituitary somatostatin receptor-2 mRNA levels. An augment in hepatic lipolysis and a reduction in serum levels of non-esterified fatty acids (NEFA) and triglycerides were found in leptin-treated rats. These rats experienced a rise in lipogenic-related factors and normalization of serum levels of NEFA and triglycerides after insulin treatment. These results suggest that an increase in insulin in leptin-treated rats can act on the hepatic lipid metabolism through activation of the GH axis.

Artocarpus altilis leaf extract protects pancreatic islets and improves glycemic control in alloxan-induced diabetic rats

The antioxidant capacity of Artocarpus altilis leaf extract may offer protection against stress oxidative-induced damage to pancreatic cells. This study aimed to examine the effect of Artocarpus leaf extract on pancreatic islets structure, blood glucose (BG), and insulin serum levels in alloxan-induced diabetic rats. Diabetes mellitus was induced in rats with an intraperitoneal injection of alloxan (155 mg/kg). Rats’ BG levels were measured daily. Only rats with BG >250 mg/dL (n = 25) proceeded to receive different treatments: placebo, Artocarpus leaf extract at the dose of 100, 200, or 400 mg/kg, or insulin 6 IU/200g. All treatments were administered daily for 14 days before blood and pancreatic tissue samples were collected. Five healthy rats (n = 5) were included to serve as normal controls. The result shows that alloxan-induced atrophy of pancreatic islets and Artocarpus leaf extract administration at all given doses reduced the severity of pancreatic islet’s atrophy. However, only at 400 mg/kg dose, Artocarpus leaf extract significantly reduced rats’ BG level (P < 0.05), similar to that of insulin-treated rats. Artocarpus leaf extract, especially at 100 and 400 mg/kg doses, also improved insulin serum levels compared with placebo treatment (P < 0.05). In conclusion, Artocarpus leaf extract protected rats’ pancreatic islets against alloxan-induced damage. This protection could improve the BG and insulin serum levels in Artocarpus-treated rats.

Perturbed ovarian and uterine glucocorticoid receptor signaling accompanies the balanced regulation of mitochondrial function and NFκB-mediated inflammation under conditions of hyperandrogenism and insulin resistance

AimThis study aimed to determine whether glucocorticoid receptor (GR) signaling, mitochondrial function, and local inflammation in the ovary and uterus are intrinsically different in rats with hyperandrogenism and insulin resistance compared to controls. Main methodsFemale Sprague Dawley rats were exposed to daily injections of human chorionic gonadotropin and/or insulin. Key findingsIn both the ovary and the uterus, decreased expression of the two GR isoforms was concurrent with increased expression of Fkbp51 but not Fkbp52 mRNA in hCG + insulin-treated rats. However, these rats exhibited contrasting regulation of Hsd11b1 and Hsd11b2 mRNAs in the two tissues. Further, the expression of several oxidative phosphorylation-related proteins decreased in the ovary and uterus following hCG and insulin stimulation, in contrast to increased expression of many genes involved in mitochondrial function and homeostasis. Additionally, hCG + insulin-treated rats showed increased expression of ovarian and uterine NFκB signaling proteins and Tnfaip3 mRNA. The mRNA expression of Il1b, Il6, and Mmp2 was decreased in both tissues, while the mRNA expression of Tnfa, Ccl2, Ccl5, and Mmp3 was increased in the uterus. Ovaries and uteri from animals co-treated with hCG and insulin showed increased collagen deposition compared to controls. SignificanceOur observations suggest that hyperandrogenism and insulin resistance disrupt ovarian and uterine GR activation and trigger compensatory or adaptive effects for mitochondrial homeostasis, allowing tissue-level maintenance of mitochondrial function in order to limit ovarian and uterine dysfunction. Our study also suggests that hyperandrogenism and insulin resistance activate NFκB signaling resulting in aberrant regulation of inflammation-related gene expression.

Involvement of neuropeptide CART in the central effects of insulin on feeding and body weight

While insulin secreted from pancreas plays a pivotal role in the control of glucose homeostasis, it also interacts with hypothalamic sites and negatively influences the energy balance. The present study was undertaken to reveal the functional interaction between cocaine- and amphetamine-regulated transcript (CART), a well-known anorexic peptide, and insulin within the framework of hypothalamus in the regulation of feeding behavior and body weight. Insulin was administered daily by intracerebroventricular (icv) route, alone or in combination with CART (icv) for a period of seven days. Immediately thereafter, preweighed food was offered to the animals at the commencement of the dark phase. The food intake and body weight were measured daily just prior to next injection. Furthermore, brains of insulin-treated rats were processed for the immunohistochemical analysis of CART-containing elements in the hypothalamus. Treatment with insulin (6 mU, icv) for a period of 7 days caused a significant decrease in food intake and body weight as compared to control. Concomitant administration of CART (0.5 μg, icv) potentiated insulin-induced anorexia and weight loss. Insulin administration resulted in a significant increase in CART immunoreactivity in the hypothalamic arcuate, paraventricular, dorsomedial and ventromedial nuclei. We suggest that increased CART contents in the hypothalamus may be causally linked with anorexia and weight loss induced by insulin.

Effect of Long-term Administration of Oral Magnesium Sulfate and Insulin to Reduce Streptozotocin-Induced Hyperglycemia in Rats: the Role of Akt2 and IRS1 Gene Expressions.

The effects of long-term oral administration of magnesium sulfate and insulin on hyperglycemia were investigated using Akt2 and IRS1 gene expression methods in streptozotocin-induced diabetic rats. Fifty rats were randomly divided into five experimental groups: 1, non-diabetic control (NDC); 2, Mg2+-treated non-diabetic control (Mg-NDC); 3, chronic diabetic (CD); 4, Mg2+-treated chronic diabetic (Mg-CD); and 5, insulin-treated chronic diabetic (Ins-CD). Streptozotocin was used to induce diabetes. The Mg-CD and Mg-NDC groups received 10g/l of MgSO4 added to drinking water. The Ins-CD group received 2.5U/kg of insulin twice a day. Blood glucose level and body weight were measured every week. The intraperitoneal glucose tolerance test (IPGTT) was performed after 16weeks. MgSO4 administration improved the blood glucose level and IPGTT. It also increased Akt2 and IRS1 genes as well as protein expression. Insulin lowered the blood glucose level and increased IRS1 gene and protein expression, but did not affect Akt2 gene and protein expression. Glucose reduction after Mg therapy may be mediated, at least partially, via IRS1 and Akt2 genes and protein stimulation. In insulin-treated rats, insulin resistance was not significant due to the absence of Akt2 gene expression.

Insulin-responsive autonomic neurons in rat medulla oblongata.

Low blood glucose activates brainstem adrenergic and cholinergic neurons, driving adrenaline secretion from the adrenal medulla and glucagon release from the pancreas. Despite their roles in maintaining glucose homeostasis, the distributions of insulin-responsive adrenergic and cholinergic neurons in the medulla are unknown. We fasted rats overnight and gave them insulin (10 U/kg i.p.) or saline after 2 weeks of handling. Blood samples were collected before injection and before perfusion at 90 min. We immunoperoxidase-stained transverse sections of perfused medulla to show Fos plus either phenylethanolamine N-methyltransferase (PNMT) or choline acetyltransferase (ChAT). Insulin injection lowered blood glucose from 4.9 ± 0.3 mmol/L to 1.7 ± 0.2 mmol/L (mean ± SEM; n = 6); saline injection had no effect. In insulin-treated rats, many PNMT-immunoreactive C1 neurons had Fos-immunoreactive nuclei, with the proportion of activated neurons being highest in the caudal part of the C1 column. In the rostral ventrolateral medulla, 33.3% ± 1.4% (n = 8) of C1 neurons were Fos-positive. Insulin also induced Fos in 47.2% ± 2.0% (n = 5) of dorsal medullary C3 neurons and in some C2 neurons. In the dorsal motor nucleus of the vagus (DMV), insulin evoked Fos in many ChAT-positive neurons. Activated neurons were concentrated in the medial and middle regions of the DMV beneath and just rostral to the area postrema. In control rats, very few C1, C2, or C3 neurons and no DMV neurons were Fos-positive. The high numbers of PNMT-immunoreactive and ChAT-immunoreactive neurons that express Fos after insulin treatment reinforce the importance of these neurons in the central response to a decrease in glucose bioavailability.

Lateral but not Medial Hypothalamic AMPK Activation Occurs at the Hypoglycemic Nadir in Insulin-injected Male Rats: Impact of Caudal Dorsomedial Hindbrain Catecholamine Signaling

The hypothalamic energy sensor adenosine 5′-monophosphate-activated protein kinase (AMPK), an important regulator of counter-regulatory responses to hypoglycemia, responds to pharmacological manipulation of hindbrain AMPK activity. Dorsomedial hindbrain A2 noradrenergic neurons express hypoglycemia-sensitive metabolo-sensory biomarkers, including AMPK. Here, adult male rats were pretreated by intra-caudal fourth ventricular administration of the selective neurotoxin 6-hydroxydopamine (6-OHDA) to determine if catecholamine signaling from the aforesaid site governs hypothalamic AMPK activation during insulin-induced hypoglycemia (IIH). Micropunched arcuate (ARH), ventromedial (VMH), paraventricular (PVH), dorsomedial (DMH) nuclei and lateral hypothalamic area (LHA) tissues were obtained at the neutral protamine Hagedorn insulin-induced hypoglycemic nadir, coincident with A2 AMPK activation, for Western blot analysis of AMPK, phospho-AMPK (pAMPK), and relevant metabolic neuropeptides. ARH, VMH, LHA, and DMH norepinephrine levels were altered according to insulin dose; 6-OHDA-mediated reversal of these responses was site-specific. IIH elevated LHA and reduced VMH pAMPK protein, profiles that were respectively unchanged or increased by 6-OHDA. PVH and ARH pAMPK was resistant to IIH, but augmented in ARH of neurotoxin- plus insulin-treated rats. ARH neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) proteins were correspondingly increased or refractory to IIH; 6-OHDA pretreatment normalized NPY and elevated POMC expression after insulin injection. Results demonstrate site-specific bi-directional adjustments in hypothalamic AMPK reactivity to hypoglycemia. Intensification of ARH/VMH pAMPK by 6-OHDA implies dorsomedial hindbrain improvement of energy balance in those sites during IIH. Neurotoxin-mediated augmentation versus suppression of basal catabolic (ARH POMC/VMH steroidogenic factor-1) or IIH-associated anabolic (ARH NPY) neuropeptide profiles, respectively, may involve local AMPK-dependent against independent mechanisms.

Region-dependent effects of diabetes and insulin-replacement on neuronal nitric oxide synthase- and heme oxygenase-immunoreactive submucous neurons.

AIMTo investigate the intestinal segment-specific effects of diabetes and insulin replacement on the density of different subpopulations of submucous neurons.METHODSTen weeks after the onset of type 1 diabetes samples were taken from the duodenum, ileum and colon of streptozotocin-induce diabetic, insulin-treated diabetic and sex- and age-matched control rats. Whole-mount preparations of submucous plexus were prepared from the different gut segments for quantitative fluorescent immunohistochemistry. The following double-immunostainings were performed: neuronal nitric oxide synthase (nNOS) and HuC/D, heme oxygenase (HO) 1 and peripherin, as well as HO2 and peripherin. The density of nNOS-, HO1- and HO2-immunoreactive (IR) neurons was determined as a percentage of the total number of submucous neurons.RESULTSThe total number of submucous neurons and the proportion of nNOS-, HO1- and HO2-IR subpopulations were not affected in the duodenal ganglia of control, diabetic and insulin-treated rats. While the total neuronal number did not change in either the ileum or the colon, the density of nitrergic neurons exhibited a 2- and 3-fold increase in the diabetic ileum and colon, respectively, which was further enhanced after insulin replacement. The presence of HO1- and HO2-IR submucous neurons was robust in the colon of controls (38.4%-50.8%), whereas it was significantly lower in the small intestinal segments (0.0%-4.2%, P < 0.0001). Under pathophysiological conditions the only alteration detected was an increase in the ileum and a decrease in the colon of the proportion of HO-IR neurons in insulin-treated diabetic animals.CONCLUSIONDiabetes and immediate insulin replacement induce the most pronounced region-specific alterations of nNOS-, HO1- and HO2-IR submucous neuronal density in the distal parts of the gut.

Assessment of SGLT2 inhibitors on kidney morphology and albumin excretion in rats with experimental diabetes mellitus type 1

Background. Recently, as the therapy of diabetes mellitus, have been approved several members of a new class of drugs – SGLT2 inhibitors. SGLT2 inhibitors reduce glycemia and normalize renal perfusion. However renoprotective effect is still the subject of research.The aim of this study was to evaluate the effect of SGLT2 inhibitors on renal tissue of rats with experimental type 1 diabetes (DM).Materials and methods. This study was conducted on 22 white male rats at the age of 10 months with streptozotocin - induced diabetes. For the selection of the rats in the experiment were evaluated glycemia. The criteria for diabetes was blood glucose levels &gt; 7 mmol / L and / or a positive glucose tolerance test. Rats were divided into 3 groups: 1 group (healthy control) - a group of 10 animals without DM, 2 group - (diabetic control) 6 animals with DM receiving insulin NPH; 3 -6 animals with DM receiving insulin NPH and dapagliflozin 0.1 mg / kg for 4 weeks. At the end of the study, animals were kept in metabolic cages and 24 hour urine was collected for estimation of albuminuria. Next animals were removed from the experiment, and kidney tissue was sampled for morphological evaluation. Sections were stained with haematoxylin and eosin (H&amp;E), and PAS reaction stains for histopathological examination. Statistical significance of differences was assessed and linkages by standard methods of nonparametric statistics.Results. Sections of kidney from control group had classical structure of renal tissue. In contrast, histological sections from rats treated with insulin had showed cortical glomerulosclerosis, proliferation of mesangial cells and narrowed Bowman's spaces. In most of the proximal convoluted tubules was observed excessive hypertrophy, vacuolization and pyknotic nuclei. The kidneys of rats treated with dapagliflozin and insulin had a lower severity of degenerative processes compared with a group of rats treated with insulin. Many glomeruli had increased cellularity with normal Bowman’s spaces, while in the proximal convoluted tubules was observed weakly pronounced vacuolization and pyknotic nuclei and some less hypertrophy of tubular epithelium. Kidney sections of insulin-treated rats were showed signs of diffuse expansion of mesangial area with proliferation of mesangial cells and its PAS-positive matrix. While signs of mesangial expansion were absent in the dapagliflozin group. Analysis of the degree of glomerulosclerosis data was showed significant differences between the group of rats treated with dapagliflozin and insulin and the group of rats treated with insulin - 0.5 (0.4 - 0.6) and 1.1 (1.0 - 1.2), respectively ( p = 0.005). Furthermore, it revealed significant difference in percentage of mesangial area between group of rats treated with dapagliflozin and insulin and the group of rats treated with insulin - 28% (23 - 32) and 37% (33 - 41), respectively (p = 0.0082). Insulin-treated rats were showed significantly higher level of albuminuria compared with dapagliflozin-treated rats - 91.8 mg / 24h. (74.1 - 108.5) and 50.9 mg / 24 hr (41.3 - 60.2), respectively, (p = 0.012).Conclusions. Administration of dapagliflozin slows the progression of glomerulosclerosis and reduces the degree of its severity and the level of albuminuria, which may suggest a renoprotective properties.

The glucoregulatory response to high-intensity aerobic exercise following training in rats with insulin-treated type 1 diabetes mellitus.

An acute bout of exercise elicits a rapid, potentially deleterious, reduction in blood glucose in patients with type 1 diabetes mellitus (T1DM). In the current study, we examined whether a 10-week aerobic training program could alleviate the rapid exercise-associated reduction in blood glucose through changes in the glucoregulatory hormonal response or increased hepatic glycogen storage in an insulin-treated rat model of T1DM. Thirty-two male Sprague-Dawley rats were divided evenly into 4 groups: non-T1DM sedentary (C) (n = 8), non-T1DM exercised (CX) (n = 8), T1DM sedentary (D) (n = 8), and T1DM exercised (DX) (n = 8). Exercise training consisted of treadmill running for 5 days/week (1 h, 27 m/min, 6% grade) for 10 weeks. T1DM was induced by multiple streptozotocin injections (20 mg/kg) followed by implantation of subcutaneous insulin pellets. At week 1, an acute exercise bout led to a significant reduction in blood glucose in DX (p < 0.05), whereas CX exhibited an increase in blood glucose (p < 0.05). During acute exercise, serum epinephrine was increased in both DX and CX (p < 0.05), whereas serum glucagon was increased during recovery only in CX (p < 0.01). Following aerobic training in DX, the exercise-mediated reduction in blood glucose remained; however, serum glucagon increased to the same extent as in CX (p < 0.05). DX exhibited significantly less hepatic glycogen (p < 0.001) despite elevations in glycogenic proteins in the liver (p < 0.05). Elevated serum epinephrine and decreased hepatic adrenergic receptor expression were also evident in DX (p < 0.05). In summary, despite aerobic training in DX, abrupt blood glucose reductions and hepatic glycogen deficiencies were evident. These data suggest that sympathetic overactivity may contribute to deficiencies in hepatic glycogen storage.

Renal Effects of Glucose Transporter 4 in Nω‐nitro‐L‐arginine /High Salt Induced‐Hypertensive Rats

The aim of study was to determine the renal effects of glucose transporter 4 (GLUT4) in a hypertensive nephropathy rat model. GLUT4 has been implicated in insulin resistance and hypertension in several animal models, however its role in hypertensive nephropathy still remain unclear. Hypertensive nephropathy was induced by Nω‐nitro‐L‐arginine (L‐NNA), a nitric oxide (NO) synthase inhibitor, 100 mg/ml in drinking water and high salt (HS) diet (4 % NaCl), for 15 days in the presence of insulin, a GLUT 4 agonist (1 U/day) and indinavir, a GLUT4 inhibitor (80 mg/kg/day). Decreased basal renal medullary and cortical blood flow was enhanced in LNNA/HS/indinavir group (p&lt;0.01) but attenuated (p&lt;0.05) by insulin. Proteinuria was increased (p&lt;0.01) in LNNA/HS/indinavir group but attenuated (p&lt;0.01) by insulin. Insulin‐treated rats decreased urine NO (p&lt;0.01) and urine Na2+ (p&lt;0.01) compared to other treated animals. In indinavir‐treated animals, urine Na2+ was increased by benzamil, an epithelial sodium channel (ENaC) inhibitor (p&lt;0.01) and hydrochlorothiazide, a sodium/chloride co‐transporter (NCC) inhibitor (p&lt;0.05). GLUT4 exerts a renoprotective role which may be related to increase NO production. The antinatriuretic effects of GLUT4 appear to be due to enhancement of ion transport activity of ENaC and NCC at the renal tubules.This study was supported by National Institutes of Health grant HL03674.

Experimental diabetes and the epididymis of Wistar rats: The protective effects of Anacardium occidentale (Linn.)

Aim: The use of botanical remedies as adjunct therapies in the management of diabetes mellitus is on the increase. Anacardium occidentale has been reported in the literature to possess anti-diabetic and hypoglycemic properties. This study evaluated the effects of acute treatment of A. occidentale on blood glucose and epididymis histopathology of streptozotocin (STZ)-diabetic rats. Materials and Methods: Forty adult male Wistar rats were randomly divided into 4 experimental groups of 10 rats each. A group served as the normoglycemic control and was administered 1 ml/kg bw/day citrate buffer. Hyperglycemia was induced in 30 overnight-fasted rats with a single i.p injection of STZ (70 mg/kg bw/day). Hyperglycemia was confirmed 48 h later and thereafter allowed to stabilize for 5 days. 300 mg/kg bw/day of ethanolic extract of A. occidentale was administered orally to a group of diabetic rats (n = 10). Insulin was also administered subcutaneously at 10 I.U/kg bw/day to another group (n = 10). Another group served as the hyperglycemic control and received 1 ml of citrate buffer/kg bw/day. Treatment after a 5-day stabilization of hyperglycemia lasted for 17 days. In each group blood glucose and epididymal histology were assessed. Results: By the end of the experimental period, all hyperglycemic rats in the extract-treated group had become normoglycemic. Moreover, extract-treated rats showed improved epididymal morphology and luminal sperm aggregate within the duct comparable to normoglycemic and insulin-treated rats. Conclusion: We conclude that A. occidentale proved valuable in mitigating the detrimental effects of hyperglycemia on the epididymis.